1:100:2, vvv was added to the homogenates. After centrifugation, the aqueous phase was mixed
with 1 vol. isopropanol, and then placed at −
20°C for at least 1 h to precipitate RNA. The pellet was dissolved in TNE buffer 50 mM Tris –
HCl, pH 7.5, 10 mM EDTA, 0.5 SDS and again precipitated by adjusting the solution to 2 M
LiCl. After centrifugation, the RNA pellet was washed with 80 ethanol, dried and dissolved in
DEPC-treated water. For Northern blot analysis, total RNA 20 mg was separated on a denaturing
1.5 formaldehyde agarose gel. The RNA was transferred to Hybond N
+
membrane Amer- sham, followed by UV cross-linking. Hybridiza-
tion was performed overnight at 50°C in a solution containing 7 SDS, 50 formamide, 5×
SSC, 2 blocking reagent, 50 mM sodium phos- phate pH 7.0, and 0.1 N-lauroylsarcosine.
Membranes were washed twice with 2× SSC and 0.1 SDS at room temperature for 10 min, and
then twice with 0.1× SSC and 0.1 SDS at 65°C for 5 min. Membranes were exposed to X-ray
films.
3. Results
3
.
1
. Isolation of a cDNA clone encoding a basic class II chitinase
Two primers, N-terminus CLASS2N and C-ter- minus CLASS2C, were designed and synthesized
from the conserved regions of the class II chitinase sequences previously reported in many plant spe-
cies. The polymerase chain reaction PCR proce- dures were performed to amplify the coding region
of the class II pepper chitinase from pepper genomic DNA using the two primers synthesized.
After the PCR amplification, a distinct DNA frag- ment was visualized on an agarose gel. The size of
the DNA fragment amplified was similar to the size expected, approximately 310 bp, which corre-
sponds to the coding region of the class II chiti- nases between the two primers. The fragment was
cloned into pT7Blue cloning vector and se- quenced. The amplified fragment, named pchit
II, of the class II pepper chitinase was identified to be 308 bp data not shown. The pchit II
sequence showed 79, 76, and 71 identities to potato, tomato and tobacco class II chitinase,
respectively. The cDNA library, which was constructed from
mRNAs in pepper leaves inoculated with the avir- ulent strain Bv5-4a of X. campestris pv. 6esicato-
ria, was screened with the PCR-derived clone pchit II.
Among approximately
50 000 plaques
screened, more than 150 plaques showed positive hybridizations with the pchit II probe at high
stringency. Following plaque purification and in vivo excision of the pBluescript SK − recombi-
nant plasmid from the lZAP II, the sizes of 15 clones were determined by agarose gel elec-
trophoresis and Southern blot analysis data not shown. The two cDNA clones of ca. 1.5 and 0.4
kb, which hybridized to the pchit II probe, were sequenced. However, the 1.5-kb cDNA clone pchit
II 1-6 was confirmed to encode a putative basic chitinase of pepper in the sequence comparison by
the BLAST program. The putative basic chitinase cDNA pchit II 1-6 was designated CAChi2.
3
.
2
. Nucleotide and deduced amino acid sequences of CAChi
2
cDNA The complete nucleotide and deduced amino
acid sequences of the CAChi2 cDNA encoding a putative basic chitinase of pepper revealed that the
CAChi2 cDNA contains 1004 bp with a 759-nu- cleotide open reading frame data not shown. The
N-terminal 24 amino acids exhibit the characteris- tics of a signal peptide with a highly hydrophobic
core and characteristic amino acid composition near the cleavage site [31]. The CAChi2 does not
have cysteine-rich domains and a C-terminal ex- tension signal sequence, which is necessary for
vacuolar targeting of mature proteins [32].
The pepper CAChi2 open reading frame en- codes a 253-amino acid protein with a predicted
molecular mass of 27 768 Da and pI of 9.39. Molecular mass of mature CAChi2 protein is
25 161 Da. A search of protein databases with the deduced amino acid sequence using the Genetics
Computer Group BLAST program revealed that the protein encoded by CAChi2 shares significant
sequence identity with all other plant class II chitinases cDNAs. To examine similarities and
differences in individual amino acid sequence posi- tions, in Fig. 1, we aligned the deduced amino acid
sequence of CAChi2 with five chitinases from other plant species. The alignment shows that
CAChi2 has highly conserved regions in the cata- lytic domain with identical position and length, as
compared to the conserved regions in potato and tomato chitinases [12,33,34]. The high levels
of sequence identity, from 69 to 86, were found between the pepper chitinase CAChi2 and each of
class II chitinases of potato, tomato, tobacco, and petunia. In contrast, a much lower identity was
found between the pepper chitinase and other classes of plant chitinases data not shown.
A phylogenetic analysis of the 15 deduced chiti- nase amino acid sequences in Genbank was used to
compare the relationship of the chitinase encoded by pepper CAChi2 to other chitinases. As shown in
Fig. 2, well-separated branches of plant chitinases can be identified in the phylogenetic tree. The
deduced proteins of the CAChi2 chitinase cDNA clone falls into a class II chitinase group. Chitinases
of each class are clustered together. Class II chiti- nase group including CAChi2 has a high amino
acid sequence homology to class I chitinases, but sequence dissimilarities to class IV chitinases.
Fig. 1. Comparison of the amino acid sequences derived from pepper C. annuum L. CAChi2 cDNA with sequences from potato Solanum tuberosum ChtA2 accession no. U49969, [33], uncultivated tomato Lycopersicon chilense PCHIT28 accession no.
Q40114, [34], tomato Lycopersicon esculentum 26 kDa chitinase accession no. Z15141, [12], tobacco Nicotiana tabacum PR-P accession no. X51426, [35], and petunia Petunia hybrida 25.1 kDa chitinase accession no. X51427, [35]. Amino acids marked
in black boxes indicate sequence identity. Gaps introduced for optimal alignment are indicated by dashes – . Putative signal peptide cleavage site is represented by an arrow ¡. Two conserved regions present in other plant species, used for the primer
design, are labeled by I and II. The numbers at the right refer to amino acid residue positions in the respective sequence.
Fig. 2. Phylogenic analysis of the aligned amino acid sequences of pepper chitinase CAChi2 cDNA and 15 other plant chitinase genes. The other chitinases are indicated by the names of the plants of origin and their GenBankEMBL accession numbers. The
relative homologies between chitinase sequences are proportional to the horizontal length of branches.
3
.
3
. Genomic organization of pepper chitinase gene corresponding to CAChi
2
cDNA Genomic DNA was digested with the restriction
enzymes EcoRI, BamHI, and HindIII, respec- tively, for which there are no internal restriction
sites in the sequence of CAChi2 cDNA. The com- pletely digested DNA was subjected to Southern
blot hybridization with the EcoRI – XhoI insert from the isolated chitinase cDNA clone CAChi2
as a probe Fig. 3. The CAChi2 cDNA hybridized to one EcoRI band, one BamHI band and two
HindIII bands. All of these band sizes are long enough to represent intact genes, suggesting that
the CAChi2 chitinase in pepper genome is encoded by single or two copy genes.
3
.
4
. Expression of CAChi
2
mRNA upon infection with pathogens and treatment with abiotic elicitors
RNA gel blot analysis was performed to moni- tor the expression of the chitinase CAChi2 mRNA
upon infection with either X. campestris pv. 6esica- toria or P. capsici. No transcripts homologous to
CAChi2 cDNA were detected in the healthy pep- per leaves. In the compatible interactions of X.
campestris pv. 6esicatoria with pepper, accumula- tion of CAChi2 mRNA was considerably induced
in the leaves by 30 h after inoculation Fig. 4. In the incompatible interaction, the CAChi2 mRNA
was first detected 12 h after inoculation, followed by a maximum in transcript levels at 30 h. P.
capsici-infected pepper stems also showed different accumulation of CAChi2 transcripts in the com-
patible and incompatible interactions Fig. 5. In- duction of CAChi2 appeared in both interactions
1 day after inoculation. In the compatible interac-
Fig. 3. Genomic DNA gel blot analysis of the putative CAChi2 cDNA of pepper plants. Genomic DNA was di-
gested with EcoRI, BamHI, and HindIII, fractionated on 0.8 wv agarose gel, and blotted onto nylon membrane
Hybond N
+
. The membrane was hybridized with a CAChi2 cDNA probe. The sizes of the DNA molecular weight stan-
dards are indicated at the left.
Fig. 4. Northern blot analysis of chitinase CAChi2 mRNA accumulation in pepper leaf tissue during compatible and
incompatible interactions after infection by virulent strain Ds1 and avirulent strain Bv5-4a of Xanthomonas campestris
pv. 6esicatoria, respectively. A Northern blot analysis of CAChi2 mRNA at various times after inoculation. B Den-
sitometric comparison of CAChi2 mRNA levels between compatible and incompatible interactions. As a control, each
blot was hybridized with a 25S rRNA probe from Capsicum annuum. The Northern blot analysis was repeated three times
with similar results. Vertical bars represent S.E.
monate, benzothiadiazole and salicylic acid re- sulted in little accumulation of the transcripts in
the leaves Fig. 6A. Time course induction of CAChi2 transcripts by ethephon treatment is
shown in Fig. 6B. The transcript level began to increase at 18 h after treatment with ethephon,
with a maximum at 24 h. After application of ethephon, a chemical releasing gaseous ethylene,
we examined whether or not hydrochloric acid HCl and phosphonic acid H
3
PO
3
as breakdown products of ethephon when applied to pepper
plants induced transcripts of CAChi2. There was no induction of the transcript accumulation in
pepper leaves by treatment with the two chemicals data not shown. When treated with methyl jas-
Fig. 5. Northern blot analysis of chitinase CAChi2 mRNA accumulation in pepper stem tissue during compatible and
incompatible interactions after infection by virulent isolate S197 and avirulent isolate CBS178.26 of Phytophthora capsici,
respectively. A Northern blot analysis of CAChi2 mRNA at various times after inoculation. ‘W’ indicates total RNA
extracted from the wounded stem tissues 1 day after wound- ing. B Densitometric comparison of CAChi2 mRNA levels
between compatible and incompatible interactions. As a con- trol, each blot was hybridized with a 25S rRNA probe from
Capsicum annuum. The Northern blot analysis was repeated three times with similar results. Vertical bars represent S.E.
tions, CAChi2 slightly increased 2 days after inoc- ulation and then diminished after 3 days after
inoculation when pepper plants began to die due to severe P. capsici infection. In the incompatible
interaction, however, drastic increase of CAChi2 mRNA occurred 2 – 3 days after inoculation. After
4 days, the transcripts decreased to the level of 1 day after inoculation. There was no expression of
CAChi2 mRNA in the wounded stems.
The levels of expression of the CAChi2 mRNA in the pepper leaves in response to chemical elici-
tors were determined by RNA gel blot analysis, as shown in Fig. 6. Among the chemical elicitors
treated, ethephon strongly induced the expression of CAChi2 mRNA in pepper leaves 24 h after
treatment. In contrast, treatment with methyl jas-
Fig. 6. A Expression pattern of CAChi
2
mRNAs in pepper leaves treated with ethephon, methyl jasmonate,
DL
-b-amino- n-butyric acid, benzothiadiazole, and salicylic acid. B Time
course of induction of CAChi2 mRNA in pepper leaves treated with 10 mM ethephon. As a control, each blot was
hybridized with a 25S rRNA probe from C. annuum. The Northern blot analysis was repeated three times with similar
results.
CAChi2 belongs to the class II chitinase. The chitin-binding domain is known to be a major
characteristic of class I chitinase [38]. Class I and II chitinases have leader sequences and catalytic
regions as common features, whereas class II chiti- nase do not have a cysteine-rich chitin-binding
domain, a hinge region, and a C-terminal exten- sion sequence present in the class I chitinase [39].
Comparison of N-terminal amino acid sequences of basic pepper chitinases showed that the basic
CAChi2 chitinase is not identical with the basic chitinases b1 32 kDa, pI 9.0 and b2 22 kDa, pI
9.1 previously purified from pepper plants [3], indicating the presence of another chitinase iso-
form in pepper plants. Cluster analysis of the deduced peptide of CAChi2 cDNA with the align-
ments of chitinases from 23 known chitinases also confirmed that CAChi2 can be classified into the
class II chitinases Fig. 2. With a tobacco class I chitinase CHN50, it has been suggested that some
C-terminal extension sequences are necessary for targeting the protein to the vacuole [32,40] and
that sequence changes in the vacuolar targeting peptide allow a gradual transition from vacuolar
retention to secretion [40]. C-Terminal extension sequences were not found in the pepper CAChi2
chitinase, as in the petunia class II chitinase which has been found to be extracellularly located [35].
The calculated isoelectric point pI of CAChi2 is 9.39 that is not typical in the class II chitinases
known to be usually more acidic proteins [38]. Although the CAChi2 chitinase gene may encode
a basic chitinase, its localization seems to be extra- cellular, because of absence of a C-terminal exten-
sion signal. These suggestions are well supported by the findings of Kombrink et al. [41], which
showed that potato basic chitinase was targeted to the extracellular space. Our recent observation
that the activity of a basic chitinase isoform with pI 9.8, i.e. similar to that of CAChi2 chitinase, was
induced in the intercellular washing fluid of X. campestris pv. 6esicatoria-infected leaves [13] sup-
ports the possible extracellular localization of CAChi2 chitinase in pepper leaves.
The Southern blot analysis of the pepper chiti- nase gene indicated that the basic chitinase
CAChi2 is encoded by one or two genes in pepper plants Fig. 3. The fragment restricted with
EcoRI or BamHI was hybridized as a single band, whereas the fragment restricted with HindIII was
hybridized as double bands. These bands may monate,
DL
-b-amino-n-butyric acid, benzothiadia- zole or salicylic acid, the CAChi2 transcript did
not accumulate in the pepper leaves during the incubation for 48 h.
4. Discussion
